Molecular dissection of the genes involved in biosynthesis of polyketide and secondary metabolites of Aspergillus fumigatus and their significance in virulence. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. During the previous years, we have focused our attention on the molecular genetic aspects of conidial pigment biosynthesis since pigment is one of the visible components of the wall that protect conidia. We have characterized six developmentally associated genes involved in pentaketide melanin synthesis which are clustered within a 19kb fragment of A. fumigatus genomic DNA. Furthermore, we have shown that the conidial pigment synthetic pathway plays an important role in pathogenesis. In 2001-2003, we identified the product of one of the six clustered genes, AYG1, a novel protein which shortens the length of the polyketide carbon skeleton from a heptaketide to pentaketide. We have also purified the Ayg1 protein and identified its reaction mechanism and properties. During 2004-2005, we optimized the Agrobacterium tumefaciens mediated transformation (ATMT) system for A. fumigatus. ATMT has proven to be an efficient molecular tool for insertional mutagenesis as well as gene disruption by homologous recombination. Using the ATMT system, we disrupted the LaeA gene known to be a global regulator for the expression of several important toxins among which gliotoxin is considered most important for virulence of A. fumigatus. Unlike previous report on LaeA disruption by the spheroplast method, the LaeA gene disrupted via ATMT was indistinguishable from the wild type in its mycelial pigment formation. The level of gliotoxin produced by the mutant and virulence in mice, however, was significantly lower than the wild type.Unexpectedly, the LaeA disruptant was signicantly more susceptible to antimycotic antibiotics such as azoles, caspofungin and amphotericin B. These findings suggest that LaeA not only regulates the secondary metabolism but may also is be involved in the modulation of plasma membrane and cell wall.